Besylate salt of a btk inhibitor

ABSTRACT

The present invention provides a salt form, and compositions thereof, useful as an inhibitor of one or more protein kinases and which exhibits desirable characteristics for the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention claims priority to U.S. provisional applicationSer. No. 61/372,349, filed Aug. 10, 2010, the entirety of which ishereby incorporated herein by reference.

FIELD OF THE INVENTION

The present invention provides a salt form, and compositions thereof,useful as inhibitors of protein kinases.

BACKGROUND OF THE INVENTION

The search for new therapeutic agents has been greatly aided in recentyears by a better understanding of the structure of enzymes and otherbiomolecules associated with diseases. One important class of enzymesthat has been the subject of extensive study is protein kinases.

Protein kinases constitute a large family of structurally relatedenzymes that are responsible for the control of a variety of signaltransduction processes within the cell. Protein kinases are thought tohave evolved from a common ancestral gene due to the conservation oftheir structure and catalytic function. Almost all kinases contain asimilar 250-300 amino acid catalytic domain. The kinases may becategorized into families by the substrates they phosphorylate (e.g.,protein-tyrosine, protein-serine/threonine, lipids, etc.).

In general, protein kinases mediate intracellular signaling by effectinga phosphoryl transfer from a nucleoside triphosphate to a proteinacceptor that is involved in a signaling pathway. These phosphorylationevents act as molecular on/off switches that can modulate or regulatethe target protein biological function. These phosphorylation events areultimately triggered in response to a variety of extracellular and otherstimuli. Examples of such stimuli include environmental and chemicalstress signals (e.g., osmotic shock, heat shock, ultraviolet radiation,bacterial endotoxin, and H₂O₂), cytokines (e.g., interleukin-1 (IL-1)and tumor necrosis factor α (TNF-α)), and growth factors (e.g.,granulocyte macrophage-colony-stimulating factor (GM-CSF), andfibroblast growth factor (FGF)). An extracellular stimulus may affectone or more cellular responses related to cell growth, migration,differentiation, secretion of hormones, activation of transcriptionfactors, muscle contraction, glucose metabolism, control of proteinsynthesis, and regulation of the cell cycle.

Many diseases are associated with abnormal cellular responses triggeredby protein kinase-mediated events as described above. These diseasesinclude, but are not limited to, autoimmune diseases, inflammatorydiseases, bone diseases, metabolic diseases, neurological andneurodegenerative diseases, cancer, cardiovascular diseases, allergiesand asthma, Alzheimer's disease, and hormone-related diseases.Accordingly, there remains a need to find protein kinase inhibitorsuseful as therapeutic agents.

SUMMARY OF THE INVENTION

It has now been found that the novel salt form of the present invention,and compositions thereof, is useful as an inhibitor of one or moreprotein kinases and exhibits desirable characteristics for the same. Ingeneral, this salt form, and pharmaceutically acceptable compositionsthereof, is useful for treating or lessening the severity of a varietyof diseases or disorders as described in detail herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the FT-Raman spectrum (3400-100 cm⁻¹) of Compound 2 FormP1.

FIG. 2 depicts the PXRD pattern of Compound 2 Form P1.

FIG. 3 depicts the TG-FTIR of Compound 2 Form P1.

FIG. 4 depicts the DSC thermogram of Compound 2 Form P1 showing coolingstep 2.

FIG. 5 depicts the DSC thermogram of Compound 2 Form P1 showing thefour-step heating and cooling process.

FIG. 6 depicts the PXRD pattern of Compound 2 Form P22 compared to thePXRD pattern of Compound 2 Form P1.

FIG. 7 depicts the FT-Raman spectrum (3400-100 cm⁻¹) of Compound 2 FormP22.

FIG. 8 depicts the TG-FTIR of Compound 2 Form P22.

FIG. 9 depicts the ¹H NMR of Compound 2 Form P22 prepared according toExample 3, infra.

FIG. 10 depicts the DSC thermogram of Compound 2 Form P22

DETAILED DESCRIPTION OF THE INVENTION General Description of CertainAspects of the Invention

United States published patent application number US 20100029610,published Feb. 4, 2010 (“the '610 publication,” the entirety of which ishereby incorporated herein by reference), describes certain2,4-disubstituted pyrimidine compounds which covalently and irreversiblyinhibit activity of one or more protein kinases, including Bruton'styrosine kinase (“BTK”), a member of TEC-kinases. Such compounds includecompound 1:

Compound 1(N-(3-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide)is designated as compound number 1-182 and the synthesis of compound 1is described in detail at Example 20 of the '610 publication.

Compound 1 is active in a variety of assays and therapeutic modelsdemonstrating covalent, irreversible inhibition of BTK (in enzymatic andcellular assays). Notably, compound 1 was found to inhibit B-cellproliferation both in vitro and in vivo. Accordingly, compound 1 isuseful for treating one or more disorders associated with activity ofBTK.

It would be desirable to provide a salt form of compound 1 that, ascompared to compound 1, imparts characteristics such as improved aqueoussolubility, stability and ease of formulation. Accordingly, the presentinvention provides the besylate salt of compound 1.

According to one embodiment, the present invention provides a besylatesalt of compound 1, represented by compound 2:

It will be appreciated by one of ordinary skill in the art that thebenzensulfonic acid and compound 1 are ionically bonded to form compound2. It is contemplated that compound 2 can exist in a variety of physicalforms. For example, compound 2 can be in solution, suspension, or insolid form. In certain embodiments, compound 2 is in solid form. Whencompound 2 is in solid form, said compound may be amorphous,crystalline, or a mixture thereof. Exemplary solid forms are describedin more detail below.

In other embodiments, the present invention provides compound 2substantially free of impurities. As used herein, the term“substantially free of impurities” means that the compound contains nosignificant amount of extraneous matter. Such extraneous matter mayinclude excess benzensulfonic acid, excess compound 1, residualsolvents, or any other impurities that may result from the preparationof, and/or isolation of, compound 2. In certain embodiments, at leastabout 95% by weight of compound 2 is present. In still other embodimentsof the invention, at least about 99% by weight of compound 2 is present.

According to one embodiment, compound 2 is present in an amount of atleast about 97, 97.5, 98.0, 98.5, 99, 99.5, 99.8 weight percent wherethe percentages are based on the total weight of the composition.According to another embodiment, compound 2 contains no more than about3.0 area percent HPLC of total organic impurities and, in certainembodiments, no more than about 1.5 area percent HPLC total organicimpurities relative to the total area of the HPLC chromatogram. In otherembodiments, compound 2 contains no more than about 1.0% are percentHPLC of any single impurity; no more than about 0.6 area percent HPLC ofany single impurity, and, in certain embodiments, no more than about 0.5area percent HPLC of any single impurity, relative to the total area ofthe HPLC chromatogram.

The structure depicted for compound 2 is also meant to include alltautomeric forms of compound 2. Additionally, structures depicted hereare also meant to include compounds that differ only in the presence ofone or more isotopically enriched atoms. For example, compounds havingthe present structure except for the replacement of hydrogen bydeuterium or tritium, or the replacement of a carbon by a ¹³C- or¹⁴C-enriched carbon are within the scope of this invention.

Solid Forms of Compound 2:

It has been found that compound 2 can exist in a variety of solid forms.Such forms include polymorphs, solvates, hydrates, and amorphous. Allsuch forms are contemplated by the present invention. In certainembodiments, the present invention provides compound 2 as a mixture ofone or more solid forms selected from polymorphs, solvates, hydrates,and amorphous compound 2.

As used herein, the term “polymorph” refers to the different crystalstructures (of unsolvated forms) in which a compound can crystallize. Asused herein, the term “solvate” refers to a crystal form with either astoichiometric or non-stoichiometric amount of solvent is incorporatedinto the crystal structure. Similarly, the term “hydrate” refers to acrystal form with either a stoichiometric or non-stoichiometric amountof water is incorporated into the crystal structure.

In certain embodiments, compound 2 is a crystalline solid. In otherembodiments, compound 2 is a crystalline solid substantially free ofamorphous compound 2. As used herein, the term “substantially free ofamorphous compound 2” means that the compound contains no significantamount of amorphous compound 2. In certain embodiments, at least about95% by weight of crystalline compound 2 is present. In still otherembodiments of the invention, at least about 99% by weight ofcrystalline compound 2 is present.

In certain embodiments, compound 2 is a neat crystal form and thus doesnot have any water or solvent incorporated into the crystal structure.It has been found that compound 2 can exist in at least one distinctneat (i.e., anhydrous) crystal form, or polymorph. In some embodiments,the present invention provides a polymorphic form of Compound 2 referredto herein as Form P1. In certain embodiments, the present inventionprovides a polymorphic form of Compound 2 referred to herein as FormP22.

In certain embodiments, the present invention provides Form P1 ofCompound 2. According to one aspect, Form P1 of Compound 2 has a powderX-ray diffraction pattern substantially similar to that depicted in FIG.2. According to another embodiment, Form P1 of compound 2 ischaracterized in that it has one or more peaks in its powder X-raydiffraction pattern selected from those at about 6.21, about 9.48, andabout 13.29 degrees 2-theta. In some embodiments, Form P1 of compound 2is characterized in that it has two or more peaks in its powder X-raydiffraction pattern selected from those at about 6.21, about 9.48, andabout 13.29 degrees 2-theta. Form P1 of compound 2 is characterized inthat it has all three peaks in its powder X-ray diffraction patternselected from those at about 6.21, about 9.48, and about 13.29 degrees2-theta.

As used herein, the term “about”, when used in reference to a degree2-theta value refers to the stated value ±0.1 degree 2-theta. Methodsfor preparing Form P1 of compound 2 are described infra.

In certain embodiments, the present invention provides Form P22 ofCompound 2. According to one aspect, Form P22 of Compound 2 has a powderX-ray diffraction pattern substantially similar to that depicted in FIG.6. According to another embodiment, Form P22 of compound 2 ischaracterized in that it has one or more peaks in its powder X-raydiffraction pattern selected from those at about 7.29, about 8.38, andabout 11.12 degrees 2-theta. In some embodiments, Form P22 of compound 2is characterized in that it has two or more peaks in its powder X-raydiffraction pattern selected from those at about 7.29, about 8.38, andabout 11.12 degrees 2-theta. Form P22 of compound 2 is characterized inthat it has all three peaks in its powder X-ray diffraction patternselected from those at about 7.29, about 8.38, and about 11.12 degrees2-theta.

In some embodiments, Form P22 is characterized in a melting point of194° C. Methods for preparing Form P22 of compound 2 are describedinfra.

According to another embodiment, the present invention provides compound2 as an amorphous solid. Amorphous solids are well known to one ofordinary skill in the art and are typically prepared by such methods aslyophilization, melting, and precipitation from supercritical fluid,among others.

General Methods of Providing Compound 2:

Compound 1 is prepared according to the methods described in detail inthe '610 publication, the entirety of which is hereby incorporatedherein by reference. Compound 2 is prepared from Compound 1, accordingto the Scheme below.

As depicted in the general Scheme above, Compound 2 is prepared fromCompound 1 by combining Compound 1 with benzenesulfonic acid to form thebesylate salt thereof. Thus, another aspect of the present inventionprovides a method for preparing Compound 2:

comprising the steps of:

providing Compound 1:

combining Compound 1 with benzenesulfonic acid in a suitable solvent;and

optionally isolating Compound 2.

A suitable solvent may solubilize one or more of the reactioncomponents, or, alternatively, the suitable solvent may facilitate theagitation of a suspension of one or more of the reaction components.Examples of suitable solvents useful in the present invention are aprotic solvent, a polar aprotic solvent, or mixtures thereof. In certainembodiments, suitable solvents include an ether, an ester, an alcohol, aketone, or a mixture thereof. In certain embodiments, the suitablesolvent is methanol, ethanol, isopropanol, or acetone wherein saidsolvent is anhydrous or in combination with water or heptane. In otherembodiments, suitable solvents include tetrahydrofuran,dimethylformamide, dimethylsulfoxide, glyme, diglyme, methyl t-butylether, t-butanol, n-butanol, and acetonitrile. In another embodiment,the suitable solvent is anhydrous ethanol. In some embodiments, thesuitable solvent is MTBE.

According to another embodiment, the present invention provides a methodfor preparing Compound 2:

comprising the steps of:

combining Compound 1:

with a suitable solvent and optionally heating to form a solutionthereof;

adding benzenesulfonic acid to said solution; and

optionally isolating Compound 2.

As described generally above, Compound 1 is dissolved in a suitablesolvent, optionally with heating. In certain embodiments Compound 1 isdissolved at about 50 to about 60° C. In other embodiments, Compound 1is dissolved at about 50 to about 55° C. In still other embodiments,compound 1 is dissolved at the boiling temperature of the solvent. Inother embodiments, compound 1 is dissolved without heating.

In certain embodiments, about 1 equivalent of benzenesulfonic acid isadded to Compound 1 to afford Compound 2. In other embodiments, lessthan 1 equivalent of benzenesulfonic acid is added to compound 1 toafford Compound 2. In yet other embodiments, greater than 1 equivalentof benzenesulfonic acid is added to Compound 1 to afford Compound 2. Instill other embodiments, about 0.9 to about 1.1 equivalents ofbenzenesulfonic acid is added to Compound 1 to afford Compound 2. Inanother embodiment, about 0.99 to about 1.01 equivalents ofbenzenesulfonic acid is added to Compound 1 to afford Compound 2.

It will be appreciated that the benzenesulfonic acid may be added to themixture of Compound 1 and a suitable solvent in any suitable form. Forexample, the benzenesulfonic acid may be added in solid form or as asolution or a suspension in a suitable solvent. The suitable solvent maybe the same suitable solvent as that which is combined with Compound 1or may be a different solvent. According to one embodiment, thebenzenesulfonic acid is added in solid form. In certain embodiments, thebenzenesulfonic acid combined with a suitable solvent prior to adding toCompound 1. According to another embodiment, the benzenesulfonic acid isadded as a solution in a suitable solvent. In other embodiments, thesuitable solvent in which benzenesulfonic acid is dissolved is a polarprotic or polar aprotic solvent. Such solvents include water, alcohols,ethers, and ketones. Examples of such solvents include water, methanol,ethanol, isopropanol, acetone, tetrahydrofuran, dimethylformamide,dimethylsulfoxide, glyme, diglyme, methyl t-butyl ether, t-butanol,n-butanol, and acetonitrile. In certain embodiments the suitable solventis selected from those above and is anhydrous. According to oneembodiment, the benzenesulfonic acid is dissolved in MTBE.

In certain embodiments, the resulting mixture containing Compound 2 iscooled. In other embodiments, the mixture containing Compound 2 iscooled below 20° C.

In certain embodiments, Compound 2 precipitates from the mixture. Inanother embodiment, Compound 2 crystallizes from the mixture. In otherembodiments, Compound 2 crystallizes from solution following seeding ofthe solution (i.e., adding crystals of Compound 2 to the solution).

Crystalline Compound 2 can precipitate out of the reaction mixture, orbe generated by removal of part or all of the solvent through methodssuch as evaporation, distillation, filtration (ex. nanofiltration,ultrafiltration), reverse osmosis, absorption and reaction, by adding ananti-solvent such as heptane, by cooling or by different combinations ofthese methods.

As described generally above, Compound 2 is optionally isolated. It willbe appreciated that Compound 2 may be isolated by any suitable physicalmeans known to one of ordinary skill in the art. In certain embodiments,precipitated solid compound 2 is separated from the supernatant byfiltration. In other embodiments, precipitated solid Compound 2 isseparated from the supernatant by decanting the supernatant.

In certain embodiments, precipitated solid Compound 2 is separated fromthe supernatant by filtration.

In certain embodiments, isolated Compound 2 is dried in air. In otherembodiments isolated Compound 2 is dried under reduced pressure,optionally at elevated temperature.

Uses, Formulation and Administration Pharmaceutically AcceptableCompositions

According to another embodiment, the invention provides a compositioncomprising Compound 2 and a pharmaceutically acceptable carrier,adjuvant, or vehicle. The amount of Compound 2 in compositions of thisinvention is such that is effective to measurably inhibit a proteinkinase, particularly at least one of a TEC-kinase, or a mutant thereof,in a biological sample or in a patient. In certain embodiments, theamount of Compound 2 in compositions of this invention is such that iseffective to measurably inhibit at least one of TEC-kinase, or a mutantthereof, in a biological sample or in a patient. In certain embodiments,a composition of this invention is formulated for administration to apatient in need of such composition. In some embodiments, a compositionof this invention is formulated for oral administration to a patient.

The term “patient”, as used herein, means an animal, preferably amammal, and most preferably a human.

The term “pharmaceutically acceptable carrier, adjuvant, or vehicle”refers to a non-toxic carrier, adjuvant, or vehicle that does notdestroy the pharmacological activity of the compound with which it isformulated. Pharmaceutically acceptable carriers, adjuvants or vehiclesthat may be used in the compositions of this invention include, but arenot limited to, ion exchangers, alumina, aluminum stearate, lecithin,serum proteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

Compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered orally,intraperitoneally or intravenously. Sterile injectable forms of thecompositions of this invention may be aqueous or oleaginous suspension.These suspensions may be formulated according to techniques known in theart using suitable dispersing or wetting agents and suspending agents.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium.

For this purpose, any bland fixed oil may be employed includingsynthetic mono- or di-glycerides. Fatty acids, such as oleic acid andits glyceride derivatives are useful in the preparation of injectables,as are natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, such as carboxymethyl cellulose or similar dispersingagents that are commonly used in the formulation of pharmaceuticallyacceptable dosage forms including emulsions and suspensions. Othercommonly used surfactants, such as Tweens, Spans and other emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

Pharmaceutically acceptable compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers commonly used include lactose andcorn starch. Lubricating agents, such as magnesium stearate, are alsotypically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, pharmaceutically acceptable compositions of thisinvention may be administered in the form of suppositories for rectaladministration. These can be prepared by mixing the agent with asuitable non-irritating excipient that is solid at room temperature butliquid at rectal temperature and therefore will melt in the rectum torelease the drug. Such materials include cocoa butter, beeswax andpolyethylene glycols.

Pharmaceutically acceptable compositions of this invention may also beadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiseases of the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas ororgans.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, provided pharmaceutically acceptablecompositions may be formulated in a suitable ointment containing theactive component suspended or dissolved in one or more carriers.Carriers for topical administration of compounds of this inventioninclude, but are not limited to, mineral oil, liquid petrolatum, whitepetrolatum, propylene glycol, polyoxyethylene, polyoxypropylenecompound, emulsifying wax and water. Alternatively, providedpharmaceutically acceptable compositions can be formulated in a suitablelotion or cream containing the active components suspended or dissolvedin one or more pharmaceutically acceptable carriers. Suitable carriersinclude, but are not limited to, mineral oil, sorbitan monostearate,polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol,benzyl alcohol and water.

For ophthalmic use, provided pharmaceutically acceptable compositionsmay be formulated as micronized suspensions in isotonic, pH adjustedsterile saline, or, preferably, as solutions in isotonic, pH adjustedsterile saline, either with or without a preservative such asbenzylalkonium chloride. Alternatively, for ophthalmic uses, thepharmaceutically acceptable compositions may be formulated in anointment such as petrolatum.

Pharmaceutically acceptable compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

In some embodiments, pharmaceutically acceptable compositions of thisinvention are formulated for oral administration.

The amount of compounds of the present invention that may be combinedwith the carrier materials to produce a composition in a single dosageform will vary depending upon the host treated, the particular mode ofadministration. In certain embodiments, provided compositions areformulated so that a dosage of between 0.01-100 mg/kg body weight/day ofCompound 2 can be administered to a patient receiving thesecompositions.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of Compound 2 in the composition will also depend upon theparticular compound in the composition.

Uses of Compounds and Pharmaceutically Acceptable Compositions

Compound 2 and compositions described herein are generally useful forthe inhibition of protein kinase activity of one or more enzymes.Examples of kinases that are inhibited by Compound 2 and compositionsdescribed herein and against which the methods described herein areuseful include BTK and other TEC-kinases, including ITK, TEC, BMX andRLK, or a mutant thereof.

Bruton's tyrosine kinase (“BTK”), a member of TEC-kinases, is a keysignaling enzyme expressed in all hematopoietic cell types except Tlymphocytes and natural killer cells. BTK plays an essential role in theB-cell signaling pathway linking cell surface B-cell receptor (BCR)stimulation to downstream intracellular responses.

BTK is a key regulator of B-cell development, activation, signaling, andsurvival (Kurosaki, Curr Op Imm, 2000, 276-281; Schaeffer andSchwartzberg, Curr Op Imm 2000, 282-288). In addition, BTK plays a rolein a number of other hematopoietic cell signaling pathways, e.g., Tolllike receptor (TLR) and cytokine receptor-mediated TNF-a production inmacrophages, IgE receptor (Fc_epsilon_RI) signaling in mast cells,inhibition of Fas/APO-1 apoptotic signaling in B-lineage lymphoid cells,and collagen-stimulated platelet aggregation. See, e.g., C. A. Jeffries,et al., (2003), Journal of Biological Chemistry 278:26258-26264; N. J.Horwood, et al., (2003), The Journal of Experimental Medicine 197:1603-1611; Iwaki et al. (2005), Journal of Biological Chemistry280(48):40261-40270; Vassilev et al. (1999), Journal of BiologicalChemistry 274(3): 1646-1656, and Quek et al. (1998), Current Biology8(20): 1137-1140.

Patients with mutations in BTK have a profound block in B celldevelopment, resulting in the almost complete absence of mature Blymphocytes and plasma cells, severely reduced Ig levels and a profoundinhibition of humoral response to recall antigens (reviewed in Vihinenet al Frontiers in Bioscience 5: d917-928). Mice deficient in BTK alsohave a reduced number of peripheral B cells and greatly decreased serumlevels of IgM and IgG3. BTK deletion in mice has a profound effect on Bcell proliferation induced by anti-IgM, and inhibits immune responses tothymus-independent type II antigens (Ellmeier et al, J Exp Med 192:1611-1623 (2000)). BTK also plays a crucial role in mast cell activationthrough the high-affinity IgE receptor (Fc_epsilon_RI). BTK deficientmurine mast cells have reduced degranulation and decreased production ofproinflammatory cytokines following Fc_epsilon_RI cross-linking(Kawakami et al. Journal of Leukocyte Biology 65: 286-290).

Compound 2 is an inhibitor of BTK and therefore useful for treating oneor more disorders associated with activity of BTK. Thus, in someembodiments, the present invention provides a method for treating aBTK-mediated disorder comprising the step of administering to a patientin need thereof. Compound 2, or pharmaceutically acceptable compositionthereof.

As used herein, the term “BTK-mediated” disorders or conditions as usedherein means any disease or other deleterious condition in which BTK, ora mutant thereof, is known to play a role. Accordingly, anotherembodiment of the present invention relates to treating or lessening theseverity of one or more diseases in which BTK, or a mutant thereof, isknown to play a role. Specifically, the present invention relates to amethod of treating or lessening the severity of a disease or conditionselected from a proliferative disorder or an autoimmune disorder,wherein said method comprises administering to a patient in needthereof. Compound 2 or a composition according to the present invention.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with BTK. In some embodiments, the disease orcondition is an autoimmune disease, e.g., inflammatory bowel disease,arthritis, lupus, rheumatoid arthritis, psoriatic arthritis,osteoarthritis, Still's disease, juvenile arthritis, diabetes,myasthenia gravis, Hashimoto's thyroiditis, Ord's thyroiditis, Graves'disease, Sjogren's syndrome, multiple sclerosis, Guillain-Barresyndrome, acute disseminated encephalomyelitis, Addison's disease,opsoclonus-myoclonus syndrome, ankylosing spondylosis, antiphospholipidantibody syndrome, aplastic anemia, autoimmune hepatitis, celiacdisease, Goodpasture's syndrome, idiopathic thrombocytopenic purpura,optic neuritis, scleroderma, primary biliary cirrhosis, Reiter'ssyndrome, Takayasu's arteritis, temporal arteritis, warm autoimmunehemolytic anemia, Wegener's granulomatosis, psoriasis, alopeciauniversalis, Behcet's disease, chronic fatigue, dysautonomia,endometriosis, interstitial cystitis, neuromyotonia, scleroderma, orvulvodynia. In some embodiments, the disease or condition is ahyperproliferative disease or immunologically-mediated diseasesincluding rejection of transplanted organs or tissues and AcquiredImmunodeficiency Syndrome (AIDS, also known as HIV).

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with BTK, wherein the disease or condition isselected from heteroimmune conditions or diseases, which include, butare not limited to graft versus host disease, transplantation,transfusion, anaphylaxis, allergies (e.g., allergies to plant pollens,latex, drugs, foods, insect poisons, animal hair, animal dander, dustmites, or cockroach calyx), type I hypersensitivity, allergicconjunctivitis, allergic rhinitis, and atopic dermatitis.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with BTK, wherein the disease or condition isselected from an inflammatory disease, e.g., asthma, appendicitis,blepharitis, bronchiolitis, bronchitis, bursitis, cervicitis,cholangitis, cholecystitis, colitis, conjunctivitis, cystitis,dacryoadenitis, dermatitis, dermatomyositis, encephalitis, endocarditis,endometritis, enteritis, enterocolitis, epicondylitis, epididymitis,fasciitis, fibrositis, gastritis, gastroenteritis, hepatitis,hidradenitis suppurativa, laryngitis, mastitis, meningitis, myelitismyocarditis, myositis, nephritis, oophoritis, orchitis, osteitis,otitis, pancreatitis, parotitis, pericarditis, peritonitis, pharyngitis,pleuritis, phlebitis, pneumonitis, pneumonia, proctitis, prostatitis,pyelonephritis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis,tendonitis, tonsillitis, uveitis, vaginitis, vasculitis, or vulvitis.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with BTK, wherein the disease or condition isselected from a cancer. In one embodiment, the cancer is a B-cellproliferative disorder, e.g., diffuse large B cell lymphoma, follicularlymphoma, chronic lymphocytic lymphoma, chronic lymphocytic leukemia,acute lymphocytic leukemia, B-cell prolymphocytic leukemia,lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia, splenicmarginal zone lymphoma, multiple myeloma (also known as plasma cellmyeloma), non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmacytoma,extranodal marginal zone B cell lymphoma, nodal marginal zone B celllymphoma, mantle cell lymphoma, mediastinal (thymic) large B celllymphoma, intravascular large B cell lymphoma, primary effusionlymphoma, Burkitt lymphoma/leukemia, or lymphomatoid granulomatosis. Insome embodiments, the cancer is breast cancer, prostate cancer, orcancer of the mast cells (e.g., mastocytoma, mast cell leukemia, mastcell sarcoma, systemic mastocytosis). In one embodiment, the cancer isbone cancer. In another embodiment, the cancer is of other primaryorigin and metastasizes to the bone.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases or conditionsassociated with BTK including diseases of the bone and joints including,without limitation, rheumatoid arthritis, seronegativespondyloarthropathies (including ankylosing spondylitis, psoriaticarthritis and Reiter's disease), Behcet's disease, Sjogren's syndrome,systemic sclerosis, osteoporosis, bone cancer, and bone metastasis.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with BTK, wherein the disease or condition isselected from a thromboembolic disorder, e.g., myocardial infarct,angina pectoris, reocclusion after angioplasty, restenosis afterangioplasty, reocclusion after aortocoronary bypass, restenosis afteraortocoronary bypass, stroke, transitory ischemia, a peripheral arterialocclusive disorder, pulmonary embolism, or deep venous thrombosis.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with BTK, including infectious and noninfectiousinflammatory events and autoimmune and other inflammatory diseases.These autoimmune and inflammatory diseases, disorders, and syndromesinclude inflammatory pelvic disease, urethritis, skin sunburn,sinusitis, pneumonitis, encephalitis, meningitis, myocarditis,nephritis, osteomyelitis, myositis, hepatitis, gastritis, enteritis,dermatitis, gingivitis, appendicitis, pancreatitis, cholocystitus,agammaglobulinemia, psoriasis, allergy, Crohn's disease, irritable bowelsyndrome, ulcerative colitis, Sjogren's disease, tissue graft rejection,hyperacute rejection of transplanted organs, asthma, allergic rhinitis,chronic obstructive pulmonary disease (COPD), autoimmune polyglandulardisease (also known as autoimmune polyglandular syndrome), autoimmunealopecia, pernicious anemia, glomerulonephritis, dermatomyositis,multiple sclerosis, scleroderma, vasculitis, autoimmune hemolytic andthrombocytopenic states, Goodpasture's syndrome, atherosclerosis,Addison's disease, Parkinson's disease, Alzheimer's disease, type Idiabetes, septic shock, systemic lupus erythematosus (SLE), rheumatoidarthritis, psoriatic arthritis, juvenile arthritis, osteoarthritis,chronic idiopathic thrombocytopenic purpura, Waldenstrommacroglobulinemia, myasthenia gravis, Hashimoto's thyroiditis, atopicdermatitis, degenerative joint disease, vitiligo, autoimmunehypopituitarism, Guillain-Barre syndrome, Behcet's disease, scleraderma,mycosis fungoides, acute inflammatory responses (such as acuterespiratory distress syndrome and ischemia/reperfusion injury), andGraves' disease.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with BTK, selected from rheumatoid arthritis,multiple sclerosis, diabetes, B-cell chronic lymphocytic leukemia, acutelymphocytic leukemia, hairy cell leukemia, non-Hodgkin's lymphoma,Hodgkin's lymphoma, multiple myeloma, bone cancer, colorectal cancer,pancreatic cancer, bone metastasis, osteoporosis, irritable bowelsyndrome, Crohn's disease, lupus and renal transplant.

BTK is a member of the TEC-kinases, which members share a commoncysteine at the equivalent position of Cys481 of Btk also capable ofirreversible inhibition as described in the '610 publication.Accordingly, examples of kinases that are inhibited by Compound 2 andcompositions described herein and against which the methods describedherein are useful include additional TEC-kinases beyond BTK, includingITK, TEC, BMX and RLK, or a mutant thereof.

The activity of Compound 2 as an inhibitor of a TEC-kinase, or a mutantthereof, may be assayed in vitro, in vivo or in a cell line. In vitroassays include assays that determine inhibition of either thephosphorylation activity and/or the subsequent functional consequences,or ATPase activity of activated a TEC-kinase, or a mutant thereof.Alternate in vitro assays quantitate the ability of Compound 2 to bindto a TEC-kinase Inhibitor binding may be measured by radiolabeling theinhibitor prior to binding, isolating the inhibitor/TEC-kinase (i.e.,TEC, BTK, ITK, RLK and BMX complex and determining the amount ofradiolabel bound. Detailed conditions for assaying Compound 2 as aninhibitor of a TEC-kinase, or a mutant thereof, are set forth in detailin the '610 publication.

Protein tyrosine kinases are a class of enzymes that catalyze thetransfer of a phosphate group from ATP or GTP to a tyrosine residuelocated on a protein substrate. Receptor tyrosine kinases act totransmit signals from the outside of a cell to the inside by activatingsecondary messaging effectors via a phosphorylation event. A variety ofcellular processes are promoted by these signals, includingproliferation, carbohydrate utilization, protein synthesis,angiogenesis, cell growth, and cell survival.

As used herein, the terms “treatment,” “treat,” and “treating” refer toreversing, alleviating, delaying the onset of, or inhibiting theprogress of a disease or disorder, or one or more symptoms thereof, asdescribed herein. In some embodiments, treatment may be administeredafter one or more symptoms have developed. In other embodiments,treatment may be administered in the absence of symptoms. For example,treatment may be administered to a susceptible individual prior to theonset of symptoms (e.g., in light of a history of symptoms and/or inlight of genetic or other susceptibility factors). Treatment may also becontinued after symptoms have resolved, for example to prevent or delaytheir recurrence.

The TEC family of non-receptor tyrosine kinases, referred to herein as“TEC-kinases,” plays a central role in signaling throughantigen-receptors such as the TCR, BCR and Fc receptors (reviewed inMiller A, et al. Current Opinion in Immunology 14; 331-340 (2002).TEC-kinases are essential for T cell activation. Three members of thefamily, Itk, Rlk and, are activated downstream of antigen receptorengagement in T cells and transmit signals to downstream effectors,including PLC-γ. Combined deletion of Itk and Rlk in mice leads to aprofound inhibition of TCR responses including proliferation, cytokineproduction and immune responses to an intracellular parasite (Toxoplasmagondii) (Schaeffer et al., Science 284; 638-641 (1999)). Intracellularsignalling following TCR engagement is effected in ITK/RLK deficient Tcells; inositol triphosphate production, calcium mobilization and MAPkinase activation are all reduced. Tec-kinases are also essential for Bcell development and activation.

TEC-kinases include five family members, which are expressed primarilyin hematopoietic cells: TEC, BTK, ITK (also known as TSK and EMT), RLK(also known as TXK), and BMX (also known as ETK). Additional relatedTEC-kinases have been found in Drosophila melanogaster, zebrafish (Daniorerió), skate (Raja eglanteria), and sea urchin (Anthocidariscrassispina).

Compound 2 is an inhibitor of one of more TEC-kinases and is thereforeuseful for treating one or more disorders associated with activity ofone or more TEC-kinases. Thus, in certain embodiments, the presentinvention provides a method for treating a TEC-mediated disordercomprising the step of administering to a patient in need thereof.Compound 2, or pharmaceutically acceptable composition thereof.

The term “TEC-mediated condition”, as used herein means any disease orother deleterious condition in which TEC-kinases are known to play arole. Such conditions include those described herein and in Melcher, Met al., “The Role of TEC Family Kinases in Inflammatory Processes”,Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry, Vol. 6,No. 1, pp. 61-69 (February 2007). Accordingly, another embodiment of thepresent invention relates to treating or lessening the severity of oneor more diseases in which TEC-kinases are known to play a role.Specifically, the present invention relates to a method of treating orlessening the severity of a disease or condition selected fromautoimmune, inflammatory, proliferative, and hyperproliferative diseasesand immunologically-mediated diseases including rejection oftransplanted organs or tissues and Acquired Immunodeficiency Syndrome(AIDS) (also known as HIV), wherein said method comprises administeringto a patient in need thereof. Compound 2 or a composition thereof.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with TEC-kinases including diseases of therespiratory tract including, without limitation, reversible obstructiveairways diseases including asthma, such as bronchial, allergic,intrinsic, extrinsic and dust asthma, particularly chronic or inveterateasthma (e.g., late asthma airways hyper-responsiveness) and bronchitis.In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with TEC-kinases including those conditionscharacterized by inflammation of the nasal mucus membrane, includingacute rhinitis, allergic, atrophic rhinitis and chronic rhinitisincluding rhinitis caseosa, hypertrophic rhinitis, rhinitis purulenta,rhinitis sicca and rhinitis medicamentosa; membranous rhinitis includingcroupous, fibrinous and pseudomembranous rhinitis and scrofoulousrhinitis, seasonal rhinitis including rhinitis nervosa (hay fever) andvasomotor rhinitis, sarcoidosis, farmer's lung and related diseases,fibroid lung, and idiopathic interstitial pneumonia.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with TEC-kinases including diseases of the boneand joints including, without limitation, rheumatoid arthritis,seronegative spondyloarthropathies (including ankylosing spondylitis,psoriatic arthritis and Reiter's disease), Behcet's disease, Sjogren'ssyndrome, systemic sclerosis, osteoporosis, bone cancer, and bonemetastasis.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with TEC-kinases including diseases and disordersof the skin, including, without limitation, psoriasis, systemicsclerosis, atopical dermatitis, contact dermatitis and other eczematousdermatitis, seborrhoetic dermatitis, Lichen planus, pemphigus, bullouspemphigus, epidermolysis bullosa, urticaria, angiodermas, vasculitides,erythemas, cutaneous eosinophilias, uveitis, alopecia, greata and vernalconjunctivitis.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with TEC-kinases including diseases and disordersof the gastrointestinal tract, including, without limitation, celiacdisease, proctitis, eosinophilic gastro-enteritis, mastocytosis,pancreatitis, Crohn's disease, ulcerative colitis, food-relatedallergies which have effects remote from the gut, e.g. migraine,rhinitis and eczema.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with TEC-kinases including those diseases anddisorders of other tissues and systemic disease, including, withoutlimitation, multiple sclerosis, artherosclerosis, lupus erythematosus,systemic lupus erythematosus, Hashimoto's thyroiditis, myastheniagravis, type I diabetes, nephrotic syndrome, eosinophilia fascitis,hyper IgE syndrome, lepromatous leprosy, sezary syndrome and idiopathicthrombocytopenia purpura, restenosis following angioplasty, tumours (forexample leukemia, lymphomas, and prostate cancers), andartherosclerosis.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with TEC-kinases including allograft rejectionincluding, without limitation, acute and chronic allograft rejectionfollowing for example transplantation of kidney, heart, liver, lung,bone marrow, skin and cornea; and chronic graft versus host disease.

In some embodiments, the present invention relates to a method oftreating or lessening the severity of one or more of the diseases orconditions associated with TEC-kinases, as recited above, wherein saidmethod comprises administering to a patient in need thereof. Compound 2or a composition according to the present invention.

Compound 2 and compositions thereof, according to the method of thepresent invention, may be administered using any amount and any route ofadministration effective for treating or lessening the severity ofcancer, an autoimmune disorder, a neurodegenerative or neurologicaldisorder, schizophrenia, a bone-related disorder, liver disease, or acardiac disorder. The exact amount required will vary from subject tosubject, depending on the species, age, and general condition of thesubject, the severity of the infection, the particular agent, its modeof administration, and the like. Compound 2 and compositions of theinvention are preferably formulated in dosage unit form for ease ofadministration and uniformity of dosage. The expression “dosage unitform” as used herein refers to a physically discrete unit of agentappropriate for the patient to be treated. It will be understood,however, that the total daily usage of the compounds and compositions ofthe present invention will be decided by the attending physician withinthe scope of sound medical judgment. The specific effective dose levelfor any particular patient or organism will depend upon a variety offactors including the disorder being treated and the severity of thedisorder; the activity of the specific compound employed; the specificcomposition employed; the age, body weight, general health, sex and dietof the patient; the time of administration, route of administration, andrate of excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed, and like factors well known in the medical arts.

Pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), bucally, as an oral or nasal spray, orthe like, depending on the severity of the infection being treated. Incertain embodiments, the compounds of the invention may be administeredorally or parenterally at dosage levels of about 0.01 mg/kg to about 50mg/kg and preferably from about 1 mg/kg to about 25 mg/kg, of subjectbody weight per day, one or more times a day, to obtain the desiredtherapeutic effect.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to Compound 2, the liquiddosage forms may contain inert diluents commonly used in the art suchas, for example, water or other solvents, solubilizing agents andemulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed,groundnut, corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedor dispersed in sterile water or other sterile injectable medium priorto use.

In order to prolong the effect of Compound 2 of the present invention,it is often desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the compound thendepends upon its rate of dissolution that, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered compound form is accomplished by dissolvingor suspending the compound in an oil vehicle. Injectable depot forms aremade by forming microencapsule matrices of the compound in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofcompound to polymer and the nature of the particular polymer employed,the rate of compound release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Depot injectable formulations are also prepared by entrapping thecompound in liposomes or microemulsions that are compatible with bodytissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing Compound 2 of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, Compound 2 ismixed with at least one inert, pharmaceutically acceptable excipient orcarrier such as sodium citrate or dicalcium phosphate and/or a) fillersor extenders such as starches, lactose, sucrose, glucose, mannitol, andsilicic acid, b) binders such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c)humectants such as glycerol, d) disintegrating agents such as agar-agar,calcium carbonate, potato or tapioca starch, alginic acid, certainsilicates, and sodium carbonate, e) solution retarding agents such asparaffin, f) absorption accelerators such as quaternary ammoniumcompounds, g) wetting agents such as, for example, cetyl alcohol andglycerol monostearate, h) absorbents such as kaolin and bentonite clay,and i) lubricants such as talc, calcium stearate, magnesium stearate,solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof.In the case of capsules, tablets and pills, the dosage form may alsocomprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

Compound 2 can also be in micro-encapsulated form with one or moreexcipients as noted above. The solid dosage forms of tablets, dragees,capsules, pills, and granules can be prepared with coatings and shellssuch as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, and eye drops are also contemplatedas being within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms can be made by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

According to one embodiment, the invention relates to a method ofinhibiting protein kinase activity in a biological sample comprising thestep of contacting said biological sample with a compound of thisinvention, or a composition comprising said compound.

According to another embodiment, the invention relates to a method ofinhibiting a TEC-kinase, or a mutant thereof, activity in a biologicalsample comprising the step of contacting said biological sample withCompound 2, or a composition comprising said compound. In certainembodiments, the invention relates to a method of irreversiblyinhibiting a TEC-kinase, or a mutant thereof, activity in a biologicalsample comprising the step of contacting said biological sample withCompound 2, or a composition comprising said compound.

The term “biological sample”, as used herein, includes, withoutlimitation, cell cultures or extracts thereof; biopsied materialobtained from a mammal or extracts thereof; and blood, saliva, urine,feces, semen, tears, or other body fluids or extracts thereof.

Inhibition of protein kinase, or a protein kinase selected from aTEC-kinase, or a mutant thereof, activity in a biological sample isuseful for a variety of purposes that are known to one of skill in theart. Examples of such purposes include, but are not limited to, bloodtransfusion, organ transplantation, biological specimen storage, andbiological assays.

Another embodiment of the present invention relates to a method ofinhibiting protein kinase activity in a patient comprising the step ofadministering to said patient Compound 2, or a composition comprisingsaid compound.

According to another embodiment, the invention relates to a method ofinhibiting one or more of a TEC-kinase, or a mutant thereof, activity ina patient comprising the step of administering to said patient Compound2, or a composition comprising said compound. According to certainembodiments, the invention relates to a method of irreversiblyinhibiting one or more of a TEC-kinase, or a mutant thereof, activity ina patient comprising the step of administering to said patient Compound2, or a composition comprising said compound. In other embodiments, thepresent invention provides a method for treating a disorder mediated byone or more of a TEC-kinase, or a mutant thereof, in a patient in needthereof, comprising the step of administering to said patient Compound 2or pharmaceutically acceptable composition thereof. Such disorders aredescribed in detail herein.

All features of each of the aspects of the invention apply to all otheraspects mutatis mutandis.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

EXEMPLIFICATION

As depicted in the Examples below, in certain exemplary embodiments,compounds are prepared according to the following general procedures. Itwill be appreciated that, although the general methods depict thesynthesis of certain compounds of the present invention, the followinggeneral methods, and other methods known to one of ordinary skill in theart, can be applied to all compounds and subclasses and species of eachof these compounds, as described herein.

General Procedures

Powder X-ray diffraction patterns were obtained on a Bruker D8 Advancewith Cu—Kα radiation and LynxEye detector. The powder samples weredeposited on a zero-background polished silicon sample holder and wasrotated during measurement. Measurements performed as follows: 40 kV/40mA tube power, 0.02° 2-theta step size, 37 second step time, and 2.5-50°2-theta scanning range.

Proton Nuclear Magnetic Resonance (¹H NMR) spectra were obtained on aBruker model DPX-300 MHz NMR spectrometer. ¹H NMR spectra were recordedat 300.13 MHz, using a 30 degree excitation pulse, with a pulse delay of1 second, 16 scans. Deuterated DMSO was used as solvent.

DSC data were obtained on a Perkin Elmer DSC 7 using closed goldcrucibles. The sample was filled and dried under nitrogen. Theinstrument heated the sample at −50° C. to 250° C. at 10K/min.

TG-FTIR data were obtained using a Netzsch Thermo-Microbalance TG 209with a Bruker FT-IR Spectrometer Vector 22. Samples were measured on analuminum crucible (with microhole) under nitrogen atmosphere and heatedfrom 25-250° C. at 10K/min

Example 1 Preparation of Compound 2 (Form P1)

Compound 1 is prepared according to the method described in detail in atExample 20 of the '610 publication., the entirety of which is herebyincorporated herein by reference.

The besylate salt of Compound 1, i.e., Compound 2, was prepared asfollows. Compound 1 was added to MTBE under nitrogen to form a slurryand the mixture heated to 50-55° C. Benzenesulfonic acid, in MTBE, wasadded and the resulting mixture stirred for one hour. The mixture wascooled to 0-5° C. and allowed to stir for an hour. The resulting solidswere collected by filtration and then dried at 65-70° C. under vacuum toafford Compound 2. Characterization of the resulting materialdemonstrated that the Compound 2 was crystalline and this crystallineform referred to as Form P1.

FT-Raman spectrum for Compound 2, Form P1 is depicted in FIG. 1.

PXRD for Compound 2, Form P1 is depicted in FIG. 2. Table 1 below setsout the X-ray diffraction peaks observed for Form P22 of compound 2wherein each value is in degrees 2-theta.

TABLE 1 Observed X-ray diffraction peaks for Compound 2 (Form P1)2-Theta 6.21 9.48 11.79 13.29 14.67 16.71 17.18 17.59 19.07 19.60 19.9620.70 21.78 22.21 23.33 23.82 24.19 24.44 24.99 25.47 26.78 27.89

TG-FTIR for Compound 2, Form P1 is depicted in FIG. 3. The resultingthermogram shows the loss of about 0.9 weight % dichloromethane(residual solvent) in a step from 130-160° C.

DSC for Compound 2, Form P1 is depicted in FIG. 4 and FIG. 5.

Example 2 Solubility of Compound 2

Solubility of Compound 2, at room temperature, was measured in seventeensolvents and two solvent mixtures by manual dilution combined withvisual observation. The results are summarized in Table 2, below.

TABLE 2 Solubilities of Compound 2 Solvent Solubility mg/mL Water <1Toluene <1 TBME <1 1,4-dioxane <1 Ethyl Acetate <1 CH₂Cl₂ <1 Isopropanol<1 Anisole <1 CH₃CN ~2 Ethanol ~2 THF ~2 Acetone ~2.5 Ethylene Glycol 22< S < 17 Methanol 26 < S < 29 Benzyl alcohol  97 < S < 194 DMF >184Pyridine >234 1:1 DMF:H₂O  71 < S < 106 3:7 DMF:H₂O 16 < S < 18 — —

Example 3 Preparation of Compound 2 (Form P22)

Compound 2 (82.2 mg) was suspended in methyl ethyl ketone (6 mL) and thesuspension heated to 68° C. while adding 8 mL methyl ethyl ketone. Aclear solution was obtained and heated to 75° C. The solution was cooledto 5° C. and solvent partially evaporated to obtain a white precipitate.The resulting solid was recovered by filter centrifugation to affordform P22. The material was characterized and the results as follows:

The PXRD pattern of Compound 2 Form P22, as compared to Form P1, isdepicted in FIG. 6. The FT-Raman spectrum of Form P22 is depicted inFIG. 7. The TG-FTIR spectrum of Form P22 is depicted in FIG. 8. The ¹HNMR of Form P22 is depicted in FIG. 9 and is consistent with thestructure of Compound 2 having 1:1 ratio with its besylate salt. The DSCthermogram is depicted in FIG. 10 and shows a single endothermic eventat 193.7° C.

Table 3 below sets out the X-ray diffraction peaks observed for Form P22of Compound 2 wherein each value is in degrees 2-theta.

TABLE 3 Observed X-ray diffraction peaks for Compound 2 (Form P22)2-Theta 7.29 8.38 8.79 11.12 12.99 13.88 14.65 15.57 16.56 16.89 17.0417.69 18.35 19.10 19.43 20.00 20.53 22.08 22.38 23.21 23.66 24.34 24.6026.22 27.93 28.78 29.76

1. Compound 2:


2. The compound according to claim 1, wherein said compound is in solidform.
 3. The compound according to claim 2, wherein said compound iscrystalline.
 4. The compound according to claim 3, wherein said compoundis a crystalline solid substantially free of amorphous Compound
 2. 5.The compound according to claim 1, wherein said compound issubstantially free of impurities. 6-13. (canceled)
 14. A compositioncomprising the compound according to claim 1 and a pharmaceuticallyacceptable carrier or excipient. 15-18. (canceled)
 19. The compoundaccording to claim 2, wherein the solid form of the compound ischaracterized by at least one peak selected from the Form P1 PXRDspectra depicted in FIG.
 2. 20. The compound according to claim 19,wherein the solid form of the compound is characterized by at least twopeaks selected from the Form P1 PXRD spectra depicted in FIG.
 2. 21. Thecompound according to claim 20, wherein the solid form of the compoundis characterized by at least three peaks selected from the Form P1 PXRDspectra depicted in FIG.
 2. 22. The compound according to claim 21,wherein the solid form of the compound is characterized by at least fourpeaks selected from the Form P1 PXRD spectra depicted in FIG.
 2. 23. Thecompound according to claim 2, wherein the solid form of the compound ischaracterized by at least one peak selected from the Form P22 PXRDspectra depicted in FIG.
 6. 24. The compound according to claim 23,wherein the solid form of the compound is characterized by at least twopeaks selected from the Form P22 PXRD spectra depicted in FIG.
 6. 25.The compound according to claim 24, wherein the solid form of thecompound is characterized by at least three peaks selected from the FormP22 PXRD spectra depicted in FIG.
 6. 26. The compound according to claim25, wherein the solid form of the compound is characterized by at leastfour peaks selected from the Form P22 PXRD spectra depicted in FIG. 6.